Optical microscope which has optical modulation elements

ABSTRACT

An optical microscope is provided with an aperture element 13 and an optical modulation element 14 that creates an image having the detecting sensitivity of the phase contrast imaging technique while simultaneously realizing an image having the three-dimensional sense provided by the modulation contrast imaging technique. There are four regions (two of which may have the same transmittance value and be contiguous in portions of the optical modulation element), are provided on a single optical modulation element 14, these regions including a first light absorbing region 14a having transmittance Ta, a second light absorbing region 14b having transmittance Tb and positioned adjacent to the first light absorbing region, a first light transmitting region 14c having a transmittance Tc and positioned adjacent to the first light absorbing region 14a, and a second light transmitting region 14d having a transmittance Td, wherein Ta is greater than Tb and less than Tc, Ta is less than Td, Tc is greater than 0.5, and Td is greater than 0.5.

BACKGROUND OF THE INVENTION

In the case where a colorless transparent phase sample such as abiological cell is desired to be observed using an optical microscope,the structure cannot be seen by using a bright-field view imagingtechnique. However, various imaging techniques are known that make sucha phase sample visible. Examples of these are the phase contrast imagingtechnique, the modulation contrast imaging technique, the differentialinterference contrast (DIC) imaging technique, and so on.

The phase contrast imaging technique positions a ring ("ring" hereinmeans--annular shape--) slit at a pupil plane of an illuminating opticalsystem of a microscope, and arranges a phase-shifter (i.e., a layerwhich provides phase contrast) at a conjugate pupil plane of animage-forming optical system of the microscope. The relative phase delayof the light diffracted by the phase structure of the sample is only π/2radians as compared to the zero-order light. The phase-shifter ispositioned at the region where the zero-order light passes in order tonegate the phase difference between the zero-order light and thediffracting light. Thus, an image of the phase structure becomesvisible.

In general, light attenuation of an appropriate amount is provided onthe phase-shifter, and the contrast in the image is thus increased byhaving the zero-order light intensity and the light intensity of thediffracted light carefully controlled. This has the advantage that animage with distinctive contrast can be observed with high detectionsensitivity even for minute structures such as the granular shapesinside of cells. On the other hand, a disadvantage is that the ends ofthe structures are seen as shining white, due to a phenomenon calledhalo, and thus the detection of the outline of the structures becomedifficult.

On the one hand, the modulation contrast imaging technique arranges anaperture slit at a pupil plane of an illuminating optical system of themicroscope, as indicated in Japanese Patent Publication 51-128548, andarranges multiple regions with differing transmittances on an opticalmodulation element positioned at a conjugate pupil plane of animage-forming optical system of the microscope. Usually, a lightabsorbing layer is provided so that it has an appropriate transmittanceat the conjugate region to the aperture slit. A region adjacent one sideof this conjugate region is made to be a light transmitting region andthe region on the other side is made to be a shaded (i.e., lightblocking) region. In the pupil plane, the position where light from theslit passes varies depending on the structure in the sample so that thephase structure can be observed with shading. Advantages of themodulation contrast imaging technique are that an image having athree-dimensional sense is obtained and little expense is incurred inpracticing this imaging technique. Also, this imaging technique iswell-suited for manipulating cells and the like in that one can easilyview the outline of the structures with no halo. On the other hand, adisadvantage of the modulation contrast imaging technique is that thediscerning of minute structures is difficult, and that the detectionsensitivity is inferior in contrast to the phase contrast imagingtechnique. In addition, whenever the objective lens is exchanged, acomplicated operation must be done in order to align the aperture slitand the conjugate region thereto of the optical modulation element(i.e., the absorbing layer).

In the DIC imaging technique, a sample is illuminated by two polarizedlight beams having respective polarizations which are orthogonal to eachother by using a birefringent crystal. This technique allows minutestructures of the samples to be viewed as a result of the minutestructures affecting the polarization of the light. The polarized lightfrom the samples is made to interfere. The advantage of the DIC imagingtechnique is that a three-dimensional sense is rendered to the imagehaving extremely high contrast. On the other hand, a disadvantage isthat the equipment becomes expensive as a result of using thebirefringent crystals. And, in the case where the sample itself or asubstrate supporting the sample affects the polarization of light, anaccurate image can not be obtained. For example, the DIC imagingtechnique is not generally suitable for observing samples throughplastic surfaces because plastic can itself change the polarization ofthe light.

As explained above, in each prior art technique for imaging phasesamples there are disadvantages that arise in addition to theadvantages. Thus, an imaging technique is desired to avoid thedisadvantages while attaining the advantages.

Especially in the case where examining and manipulating biologicalsamples and the like are performed under a microscope, it is necessaryto detect even transparent, minute structures with a high sensitivity.Moreover, in order to perform accurate manipulation of minute objectsand the like, the outlines of the structures must be clearly visible.Also, it is important to be able to perform observations without theinfluence of the characteristic on polarization of samples orsubstrates. Further, there is a strong need to eliminate adjustingoperations on complex optical systems arising from exchanging objectivesof a microscope, as is frequently the case while observing microscopicstructures. Thus, it is desirable to be able to detect and manipulatesamples easily and efficiently.

There have been attempts in the prior art to achieve the above goals, inan inexpensive manner, by combining aspects of the prior art modulationcontrast technique and the phase contrast technique. An example of suchan attempt is given below.

In an embodiment of Japanese Patent Publication 57-178212, a slitaperture is arranged at a pupil plane in a position displaced outwardfrom the optical axis and oriented normal to the direction ofdisplacement from the optical axis. A light absorbing phase-shifterhaving an appropriate transmittance is provided on an optical modulationelement that is located at a pupil plane which is conjugate to thisaperture Also, as an another embodiment, among the regions adjoining thelight absorbing phase-shifter, the transmittance drops in at least oneof the adjoining regions in the direction outward from the optical axis.In both of the embodiments, one of the positive/negative first-orderdiffracted light components is blocked Thus, diffracted light thatcontributes to the image is allowed to pass on only one side of thelight absorbing phase-shifter which passes the zero-order light. Ingeneral, the contrast of the image using this technique drops as aresult of the diffracted light on one side region in relation to thezero-order light being entirely blocked. For example, there isconsideration of a thin flat phase object where the phase distributionis a sine wave with the period 1/p. The equiphase wave surface Φ(x) ofthe light that passed through this object can be represented as follows

    Φ(x)=A cos(2π p x)

wherein,

x is the axis direction parallel to the object, and A is the amplitudeof the phase distribution. Then, the complex amplitude distribution E(x)on the phase object can be considered:

    E(x)=exp {i Φ(x)}

For a small phase change, i.e., when A<<1, ##EQU1##

The above first term is the zero-order light, the second term is thepositive first-order light, and the third term is the negativefirst-order light. When the phase difference of π/2 radians between thezero-order versus the positive/negative first-order light is negatedusing a phase-shifter, the above equation becomes the below equivalentvalue E'(x).

    E'(x)=1+(A/2) exp(2 π i p x)+(A/2) exp(-2 πi p x)    Equation (1)

In equation (1), when the negative first order light is blocked:

    E"(x)=1+(A/2) exp(2 π i p x)                            Equation (2)

The respective intensities of equations (1) and (2) are given as I' (x)and I" (x), as indicated below.

    I'(x)=|E'(x)|.sup.2 ≅1+2 A cos(2 πp x)Equation (3)

    I"(x)=|E"(x)|.sup.2 ≅1+A cos(2 πp x)Equation (4)

Therefore, in comparing the blocking of one of the positive/negativefirst order light components from contributing to the image (as inequation (4)), to the case where there is no blocking of these lightcomponents (as in equation (3)), the contrast drops to approximatelyone-half. Thus, based on the structure indicated in Japanese PatentPublication 57-178212, even though modulation contrast imaging occurs,the advantage of a high detecting sensitivity as obtained in the phasecontrast imaging technique is not obtained.

In general, in the modulation contrast imaging technique, relative tothe direct light (i.e., the non-refracted light by a sample), thetransmittance drops in all the light that is passed to one side, since alight absorbing layer is arranged to attenuate this light. This providescontrast so as to allow a phase object to be viewed, with the contrasthaving the difference of brightness in the direction of refraction bythe sample. In Japanese Patent Publication 51-128548, an example of atransmittance distribution of the light absorbing region on an opticalmodulation element which is used by the modulation contrast imagingtechnique is shown. The region that the direct (i.e., zero order) lightpasses through on the optical modulation element is at the center. Aregion on one side adjoining this region has low transmittance In suchcases, even though there is contrast in an image of the phase object,the resolving power drops as a result of about half of the light fluxthat passes through the pupil plane being obstructed and thus notcontributing to the image. In order to avoid decreasing the resolvingpower, in general, apertures are arranged displaced outward from theoptical axis and the regions having low transmittance are narrowed. Bysaid patent, each region on the optical modulation elements can have aphase shift effect. However, also in this case, as a result of thediffracted light being obstructed as compared to the zero-order light,the efficacy of the phase contrast technique is low, just like that ofpreviously mentioned Japanese Patent Publication 57-178212.

Furthermore, in the above cases, because of having an asymmetric regionon an optical modulation element in relation to the optical axis, everytime the objective lens is exchanged, an adjusting operation must bedone in order to match the alignment of the aperture and the opticalmodulation element. Specifically, in the case where the opticalmodulation element is arranged at a pupil plane interior of theobjective lens, because the alignment of the optical modulation elementis not necessarily uniform in a fixed direction with respect to eachobjective lens when the objective lens is attached on a revolver, theaperture must be realigned to each objective lens when the objectivelens is exchanged. Also, along with changing the objective lens, theaperture must also be changed since the required aperture differs basedon the magnification of the lens. In using a microscope themagnification is frequently switched, and every time this occurs anexchange operation of the aperture is required.

In U.S. Pat. No. 4,407,569, a phase shift region and a light absorbingregion are independently prepared for selective insertion at a pupilplane of the image-forming optical system and, based on a suitableexchanging of the apertures that respectively correspond, one canselectively switch between the phase contrast image and the modulationcontrast image. Therefore, both the phase contrast effect and themodulation contrast effect cannot be realized at the same time. In oneembodiment of this patent, it is also disclosed that there are tworespective apertures, one for phase contrast imaging and one formodulation contrast imaging, on the same element. However, based on thisconstruction, there is a difficulty in simultaneously imaging using bothtechniques. The reason is that the zero-order light from the aperturefor phase contrast imaging receives a phase shift, although thezero-order light from the aperture for modulation contrast imaging doesnot have such a phase shift. Therefore, the interference effect of thezero-order light and the diffracted light is diminished and the contrastin the image is reduced. Even if the light absorbing regions formodulation contrast imaging were given a phase shift as well, theinterference effect between the zero-order light and the diffractedlight would be less than desirable as a result of expanding the regionin which the diffracted light is given a phase shift.

On the one hand, in order to control the characteristic halo in thephase contrast imaging technique, a phase contrast microscope thatemploys a light absorbing region apart from the phase shifter at a pupilplane is described in Japanese Patent Publication 8-94936. However, theobtaining of the effect of the modulation contrast by said phasecontrast microscope is not shown. In fact, because the shape of theaperture is also restricted to a ring, a modulation contrast image isnot obtained. Also, even where nothing is clearly shown specificallyconcerning the position and width, etc., of the light absorbing regionswhich are arranged on optical modulation elements, the effects of boththe phase contrast image and the modulation contrast image cannot besimultaneously obtained based on Japanese Patent Publication No.8-94936. In summary, both the three-dimensional sense of the modulationcontrast imaging technique and the high detection sensitivity of thephase contrast imaging technique were not able to be simultaneouslyattained by techniques that were suggested in the prior art.

As an example that reduces the changing operation of the apertureaccompanying the changing of the objective lens, a ring slit of Leica isgiven. As for the ring slit, the exchanging of the ring slit is notnecessary when there are changes of the respective magnificationsbecause the ring slit corresponds to magnifications of 10× to 40×.However, because the aperture shape is a ring, when there is use of ahigh magnification objective lens, the effective numerical aperture onthe illuminating side of the microscope is small, and the resolvingpower drops. Also, the halo becomes stronger and it becomes difficult todetect the outline of the structure of the samples.

On the one hand, in order to increase the resolution upon enlarging theilluminating ring slit diameter when a low magnification objective lensis used, the region where the zero-order light passes through the pupilplane of the image-forming optical system becomes larger. Because ofthis, there is a problem of diminished contrast.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to an optical microscope that employsoptical modulation elements.

A first object of the present invention is to provide an image oftransparent phase objects not only with a three-dimensional sense, as inthe modulation contrast imaging technique, but also with a high imagingsensitivity, as in the phase contrast imaging technique.

A second object of the invention is to make switching operations of theaperture elements unnecessary, when the first object is attained withthe objective lenses that cover a wide range of magnification, so thatthe observer's bothersome work is reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) illustrates the layout of components and ray paths of anoptical microscope for viewing an object in transmitted light having anilluminating optical system and an image-forming optical system that arealigned, and with aperture elements and optical modulation elementsdesigned according to the present invention.

FIG. 1(b) illustrates an aperture element, and

FIG. 1(c) illustrate the design of a corresponding optical modulationelement according to the present invention.

FIGS. 2(a)-2(h) illustrate other aperture elements and correspondingoptical modulation elements according to the present invention, with

FIGS. 2(a), 2(e) and 2(g) illustrating apertures of various shapes, and

FIGS. 2(b), 2(f) and 2(h) being the respective optical modulationelements that correspond.

FIG. 2(d) is another example of an optical modulation element that canbe used with the aperture element illustrated in FIG. 2(a), and FIG.2(c) is a blow-up of the pie-shaped section shown in dotted lines inFIG. 2(b).

FIG. 3 illustrates the layout of components and ray paths of an opticalsystem of an optical microscope for viewing an object in reflected lightwhich uses employs the present invention. The optical microscopeincludes a beam splitter, an illuminating optical system, and animage-forming optical system and additionally has an aperture elementand a corresponding optical modulation element of the present invention.

FIG. 4(a) illustrates the components and ray paths of anothermicroscope, for viewing an object in transmitted light and employing thepresent invention. The microscope includes a relay optical systemattached to the image-forming optical system. The relay optical systemallows the use of two optical modulation elements in separated pupilplanes, as illustrated.

FIG. 4(b) illustrates an optical modulation element 14 for use in pupilplane 11, and

FIG. 4(c) illustrates another optical modulation element 14' forsimultaneous use in pupil plane 11'. The relay optical system has theeffect of superposing the optical modulation elements 14, 14'.

FIG. 5(a) illustrates the layout of components and ray paths for aportion of an optical system according to another embodiment of theinvention in the case where a low magnification objective lens is used.

FIG. 5(b) illustrates an aperture element according to this embodimentof the invention for use in the low magnification optical system of FIG.5(a).

FIG. 5(c) illustrates details of the aperture element of FIG. 5(b), and

FIG. 5(d) illustrates an optical modulation element for use with theaperture element of FIG. 5(b).

FIG. 5(e) is an example of another optical modulation element for usewith a low magnification objective lens and light aperture as in FIG.2(b).

FIG. 6(a) illustrates the layout of components and ray paths for aportion of an optical system in the case where a high magnificationobjective lens is used for attaining the second object of the invention.

FIG. 6(b) illustrates an aperture element that is identical to that ofFIG. 5(b), and

FIG. 6(c) illustrates details of the aperture element of FIG. 6(b).

FIG. 6(d) illustrates an optical modulation element corresponding to theaperture element of FIG. 6(b), and

FIG. 6(e) is an example of an optical modulation element for use with alow magnification objective lens and the light aperture as in FIG. 2(b).

FIGS. 7(a)-7(c) illustrate another example in the case where a lowmagnification objective lens is used for attaining the second object ofthe present invention.

FIG. 7(a) illustrates the layout of components and ray paths for aportion of the optical system,

FIG. 7(b) illustrates an aperture element having improved performance ascompared to the aperture shown in FIGS. 5(b) and 6(b), and

FIG. 7(c) illustrates a corresponding optical modulation element.

FIGS. 8(a)-8(c) illustrate another example of an optical system in thecase where a high magnification objective lens is used for attaining thesecond object of the present invention.

FIG. 8(a) illustrates the layout of components and ray paths for aportion of the optical system,

FIG. 8(b) illustrates an aperture element that is identical to that ofFIG. 7(b), and

FIG. 8(c) illustrates a corresponding optical modulation element.

FIGS. 9(a)-9(c) illustrate a prior art modulation contrast imagingsystem, with

FIG. 9(a) showing the layout of components and ray paths for the entirestructure,

FIG. 9(b) illustrating the aperture element, and

FIG. 9(c) illustrating the optical modulation element.

FIGS. 10(a)-10(c) illustrate a portion of a prior art optical systemwhich uses a low magnification objective lens.

FIG. 10(a) shows the layout of components and ray paths for a portion ofthe optical system.

FIG. 10(b) illustrates an aperture element having a ring-shapedaperture, and

FIG. 10(c) illustrates a corresponding optical modulation element.

FIGS. 11(a)-11(c) illustrate a portion of a prior art optical systemwhich uses a high magnification objective.

FIG. 11(a) shows the layout of components and ray paths for a portion ofthe optical system,

FIG. 11(b) illustrates an aperture element having a ring-shapedaperture, and

FIG. 11(c) illustrates a corresponding optical modulation element.

DETAILED DESCRIPTION

In order to attain the first object of the invention, an opticalmicroscope of the present invention includes a light source, anilluminating optical system for illuminating the sample, an aperturewhich is arranged at the pupil plane of the illuminating optical systemor in that vicinity, an image-forming optical system that includes anobjective lens for forming a magnified image of a sample forobservation, and at least four light control regions arranged at one ormore pupil planes of the image-forming optical system. The four regionsinclude a first light absorbing region having a transmittance Ta whereone part or the entirety of a conjugating region to the aperture areincluded, a second light absorbing region having a transmittance Tbwhich is positioned adjacent to the first light absorbing region, afirst light transmitting region having a transmittance Tc which ispositioned adjacent to the first light absorbing region, and a secondlight transmitting region having a transmittance Td, wherein the regionon at least one optical modulation element has a phase shift effect, thesecond light transmitting region is positioned in relation to the secondlight absorbing region so as to be adjacent to the second lightabsorbing region on the side approximately opposite to the first lightabsorbing region, and wherein the following conditional equations aresatisfied:

    Tb<Ta<Tc                                                   Equation (5)

    Ta<Td                                                      Equation (6)

    Tc>0.5                                                     Equation (7)

    Td>0.5                                                     Equation (8)

Also, an optical microscope is provided which has a light source, anilluminating optical system for illuminating a sample, an apertureelement that is positioned at a pupil plane of the illuminating opticalsystem or in that vicinity and which includes an aperture in a shape ofan angular segment of a ring that is centered on the optical axis, animage-forming optical system that includes multiple objective lenses forforming an image of a sample having a selected magnification forobservation, and one or more optical modulation elements positioned atone or more pupil planes, each optical modulation element having atleast two light control regions that are arranged at different distancesfrom the optical axis, and the following conditional equation beingsatisfied:

    NAc>0.7 NAo                                                Equation (9)

wherein,

NAc is the numerical aperture value subtended by the region of anoptical modulation element having the smallest angular subtense from thesample surface,

NAo is the lowest value of numerical aperture of the multiple objectivelenses of the microscope, and

at least one of the light absorbing regions includes a phase plateregion (i.e., a region having a phase shift effect) which is the shapeof a ring that is centered on the optical axis, and only one saidaperture element is used in common with each objective lens.

The operation of the invention will now be described with reference tothe Figures. FIG. 1(a) shows the layout of the components of an opticalmicroscope of the present invention when in use, wherein there isprovided a light source 1, an illuminating optical system 2, a sample 3,an image-forming optical system 4, a collector lens 7, a pupil plane 8of the illuminating optical system, a condenser lens 9, an objectivelens 10, a pupil plane 11 of the image-forming optical system, animage-forming lens 12, an aperture element 13, and an optical modulationelement 14. An image of the sample is formed at 5, and 6 is the opticalaxis. The optical microscope of the present invention forms an image 5of sample 3 while using the combination of aperture element 13, which isarranged at pupil plane 8 of illuminating optical system 2 or in thatvicinity, and optical modulation element 14 which is arranged at pupilplane 11 of image-forming optical system 4 or in that vicinity.

FIG. 1(b) illustrates one example of an aperture element 13 withaperture 13a.

FIG. 9(c) illustrates one example of a prior art optical modulationelement 14 having regions 14a', 14b' and 14c'. Such an opticalmodulation element is generally used in the prior art modulationcontrast imaging technique as the corresponding optical modulationelement for the aperture 13a as shown in FIG. 1(b). When thetransmittances of the regions 14a', 14b', and 14c' are given in order asTa', Tb', and Tc', the following conditional equation is usuallysatisfied in the prior art modulation contrast imaging technique.

    Tb'<Ta'<Tc'

On the one hand, FIG. 1(c) illustrates an example of an opticalmodulation element 14, having four regions as the corresponding opticalmodulation element for the aperture 13a of FIG. 1(b) according to thepresent invention. This optical modulation element 14 has a first lightabsorbing region 14a, a second light absorbing region 14b, a first lighttransmitting region 14c, and a second light transmitting region 14d, andthe respective transmittances Ta, Tb, Tc, Td satisfy conditionalequations (5) (8).

In the modulation contrast imaging technique of the prior art, as shownin FIG. 9, the direct light from aperture 13a (FIG. 9(b)) which was notrefracted or diffracted by sample 3 passes through the interior ofregion 14a' on optical modulation element 14. Other light which wasrefracted or diffracted by the sample passes through regions 14b' or14c', which regions are adjacent to region 14a'. Given this, region 14b'has a transmittance lower than region 14a', causing the intensity of thelight passing through region 14b' to be weakened. On the other hand,region 14c' has a higher transmittance than region 14a'. Therefore,contrast forms in the image corresponding to the refractive indexdistribution of the sample. Thus, the prior art modulation contrastimaging technique is performed.

The effect of the modulation contrast imaging technique can also beobtained when the optical modulation element 14 of FIG. 1(c) is usedaccording to the present invention. In other words, in FIG. 1(c), thedirect light from the sample passes through region 14a, and by means ofregions 14b and 14c (which are also adjacent to region 14a), modulationcontrast imaging occurs even when using the optical modulation elementof the present invention. Both the modulation contrast imaging and thepresent invention can be given the effect of the phase contrast imagingby allowing region 14a or 14a' to have a phase shift.

But, in the present invention, by additionally providing a second lighttransmitting region 14d, the advantages of the phase contrast imagingtechnique can be combined with the modulation contrast imaging techniqueso as to yield improved imaging as compared to prior art imagingtechniques.

Compared to the case where a second light transmitting region 14d is notprovided, the present invention increases the phase contrast imagingefficacy. In short, by providing second light transmitting region 14d,the present invention attains a high detecting sensitivity because lightpassing through this region (being remote from the optical axis)contains the higher spatial frequency components which, rather thanbeing blocked as in the prior art, now contribute to the image. In otherwords it is the higher spatial frequency components that contain theinformation concerning minute structures on the sample, and this lightis no longer prevented from contributing to the image. Therefore, whenoptical modulation element 14 of FIG. 1(c) pertaining to the presentinvention is used, one obtains a higher phase contrast efficacy ofdetecting sensitivity than when the optical modulation element 14 ofFIG. 9(c) is used.

In Japanese Patent Publication 51-128548, it was disclosed that eachlight absorbing region used for modulation contrast imaging may have thephase shift effect. However, there is no disclosure concerning providinga second light transmitting region as is done in the present invention.And further, there is no disclosure concerning a high phase contrastefficacy being obtained as a result of providing this region.

Therefore, as per the present invention, by providing a second lighttransmitting region 14d adjoining the second light absorbing region 14b,the efficacy of both the primary modulation contrast imaging techniqueand the phase contrast imaging technique can simultaneously be realized.This enables the ends of the structures of the phase sample to beclearly observed, and an image having a three-dimensional sense whereshadows appear to have formed on the structure can be obtained. It alsoallows observation with a high detecting sensitivity of such things asminute structures, objects of low refractive indices, and the like.

In the construction of the present invention, it is desirable to providea phase shift as mentioned above at the previously mentioned first lightabsorbing region. Instead of a phase delay being applied at the firstabsorbing region, all the other regions of the optical modulationelement could instead be made to impart a phase shift. However, such anapproach causes the manufacturing of the phase-shifter to become morecomplicated.

In the construction of the present invention, the second light absorbingregion is made to be a shading (i.e., light blocking) layer, and it isdesirable for the transmittance of the first light transmitting regionand the second light transmitting region to be above 90%. This allowsthe contrast in the image to be increased even more.

Also, it is desirable for the first light absorbing region and saidsecond light absorbing region on the optical modulation element to bepositioned in a direction approximately radial (i.e., orthogonal) to theoptical axis. This is because contrast formation in the presentinvention is in a direction parallel to the alignment of the first lightabsorbing region and the second light absorbing region, and thedirection that most light is diffracted into is also the same radialdirection. Also, in relation to a direction orthogonal to the opticalaxis, it is desirable for the width Wa of the first light absorbingregion and the width Wb of the second light absorbing region to satisfythe following conditional equation:

    Wa/2<Wb<2Wa                                                Equation (10)

The most suitable resolving power and contrast can be obtained bysatisfying the above conditional equation. When Wb exceeds 2 Wa, thecontrast is low because the quantity of the diffracting light is low,and also, when Wb drops below Wa/2, the three-dimensional sense is vagueand the modulation contrast efficacy drops.

Moreover, it is desirable for there to be provided an aperture elementhaving an aperture shaped so as to be an angular segment of a ringcentered about the optical axis, and it is desirable for the opticalmodulation element to have a first light absorbing region and a secondlight absorbing region that are shaped as rings of different sizes andcentered on the optical axis. Thus, the arrangement of each region onthe optical modulation element becomes rotationally symmetric about theoptical axis, and it becomes unnecessary to adjust the alignments of theoptical modulation elements with the aperture shape. Also, even in thecase where observations with various directions of contrast in the imageare required, one may merely rotate the aperture element without anyfurther need to rotate or align the optical modulation element(s).

The manner in which the second object of the invention may be attainedwill now be explained with reference to the drawings.

FIGS. 10(a)-10(c) illustrate a portion of a prior art phase contrastimaging system using an objective lens which has the smallest numericalaperture among multiple objective lenses in use. FIG. 10(a) shows thelayout of components and ray paths for a portion of the construction,FIG. 10(b) illustrates the aperture element, and FIG. 10(c) illustratesthe corresponding optical modulation element. The portion of theconstruction illustrated in FIG. 10(a) is from pupil plane 8 of theilluminating optical system to pupil plane 11 of the image-formingoptical system when objective lens 10L is used which has the smallestnumerical aperture (and generally the lowest magnification) amongmultiple objective lenses in use. At pupil plane 8 of the illuminatingoptical system, the region where light flux 16L passes through adiameter corresponding to the numerical aperture of objective lens 10Lis the region of efficacy. More specifically, because the numericalaperture of low magnification objective lenses is small, only the regionin the vicinity of the optical axis at or near the pupil plane of theilluminating optical system becomes effective. This figure shows lightflux 16L using a dashed line.

On the one hand, FIGS. 11(a)-11(c) illustrate the same prior art opticalsystem as in FIG. 10, but when a high magnification objective lens 10His in use. Because the numerical aperture of a high magnificationobjective lens is high, at the pupil plane 8 of the illuminating opticalsystem, the region where a light flux 16H passes is larger than when thelow magnification objective lens is used.

In a phase contrast imaging technique of the prior art, the inner andouter diameters of the ring-shaped, phase-shifter which is arranged atthe pupil plane 11 of the image-forming optical system are sized to thenumerical aperture of the objective lens so that the most appropriatephase contrast efficacy can be obtained in relation to the pupildiameter of the objective lens in use. Because of this, the outer andinner ring diameters corresponding to the light absorbing, phase-shifteralso generally differ according to the numerical aperture of theobjective lens in use. FIG. 10(b) and FIG. 11(b) respectively illustratethe ring slits of aperture elements corresponding to when a lowmagnification objective lens versus when a high magnification objectivelens is used. FIG. 10(c) and FIG. 11(c) illustrate corresponding opticalmodulation elements (phase plates) 14L and 14H which are arranged at thepupil plane of the objective lens.

Therefore, whenever there is an exchanging of an objective lens tochange the viewing magnification, the ring slit must also be exchanged.In cases where there is frequent switching of the magnification whileobserving a sample, the exchanging of the ring slit becomes extremelyburdensome. This is not limited to the phase contrast imaging technique,and the modulation contrast imaging technique and the like all have thiscommon problem

FIGS. 5(a)-5(c) and 6(a)-6(c) illustrate the manner in which the firstand the second objects of the invention are attained. FIG. 5(b) and FIG.6(b) illustrate the identical aperture 13a that is arranged at pupilplane 8, in the case of objective lenses of both low and highmagnification being used.

FIG. 5(d) illustrates optical modulation element 14L that is arranged atpupil plane 11 of the image-forming optical system in the case where thesmallest numerical aperture objective lens is used (of multipleobjective lenses that are available for use). FIG. 6(d) illustrates theoptical modulation element 14H that is for use with the same aperture asillustrated in FIG. 5(b) or FIG. 6(b), but is used in the case where theobjective lens to be used is one having a generally high magnificationand a large numerical aperture.

In FIG. 5(d) and FIG. 6(d), a ring phase-shifter 14e having a diametercorresponding to aperture 13a is arranged. As stated previously, acommon aperture element 13a is used both in the case of a lowmagnification objective lens (FIG. 5(b)) or a high magnificationobjective lens (FIG. 6(b)) and thus an exchanging operation of theapertures accompanying the switching of the objective lens becomescompletely unnecessary.

In the case where the same aperture element is used with each ofmultiple objective lenses of different numerical aperture, the region onthe optical modulation element that the zero-order light passes throughbecomes more narrow when a higher magnification objective lens is used,and there is no assurance that a sufficient quantity of light in theimage will be maintained. Because of this, it is desirable to make thearea of the aperture as large as possible in relation to a highmagnification objective. However, this may cause a problem when a lowmagnification lens replaces the high magnification lens, in that thetransmitting region for the zero-order light on the optical modulationelement may conversely become too large. In order to use a commonaperture with objective lenses that cover as wide a range ofmagnification as possible, aperture 13a of FIG. 5(b) or FIG. 6(b) mayinstead be arranged at a position separated as far as possible from theoptical axis within the region 16L. This satisfies Equation (9). (Thisalternative arrangement of aperture 13a will be illustrated in laterfigures.)

In such a construction, in the case where the shape of the aperture ismade to be a ring as in the usual phase contrast imaging technique, whena low magnification objective lens 10L is used, the region of thezero-order light on optical modulation element 14L becomes larger thanthat as illustrated in FIG. 10(c), and it becomes impossible to maintainthe balance of the zero-order light and the diffracting light so as toachieve the most suitable phase contrast efficacy Therefore, aperture13a is made to be an angular segment of a ring (as illustrated in FIGS.5(b) and 6(b)).

Although the shape of the aperture is asymmetric in relation to theoptical axis, since the region on the optical modulation elements isrotationally symmetric, no adjusting of the alignments of the aperturesand the optical modulation elements is required.

Because the aperture is made to illuminate the sample from an off-axisposition (the oblique illumination), by making it a shape that covers anangular segment of a circular ring, the resolving power and the contrastcan be more increased on observation both with low magnification andalso with high magnification, than those in the case where a ring slitis used that is made most suitable for low magnification use. Also,based on Equation (9) being satisfied, observation at a high resolvingpower at each magnification, and an objective lens of highermagnification can be used with one common aperture. In the case whereEquation (9) is not satisfied, the effective numerical aperture becomestoo small, especially when there is a high magnification objective lensused for observation, and the resolving power drops.

Also, when the smallest numerical aperture objective lens of thoseavailable for use is employed, the conjugating regions to the apertureare positioned at the most peripheral edge of the pupil of theimage-forming optical system or in that vicinity And, on the opticalmodulation elements, the light absorbing regions which include theregions conjugating to the aperture can be arranged as rings centered onthe optical axis. A selected phase delay can also be provided at thelight absorbing regions. In this case, in relation to the objectivelenses with the smallest numerical aperture, apertures are positionedwith the highest resolving power. Such an aperture is shown in FIG.7(b). With objective lenses having the smallest numerical aperture, amodulation contrast efficacy can be obtained in the image.

Also, if the apertures are structured so that rotation about the opticalaxis is possible, the direction of contrast formation in the imagechanges. And, in case it is desired to change the direction of contrastformation in the image, adjustment at all of the optical modulationelements is not necessary.

Based on the above means, since equivalent apertures can be used inrelation to multiple objective lenses, an exchanging operation of theapertures becomes unnecessary even when there is an exchanging of theobjective lens. Moreover, when combined with the means for attaining thefirst object of the invention, an image can be attained thatsimultaneously provides a high imaging sensitivity, as in the phasecontrast imaging technique, as well as a clear outline with athree-dimensional sense, as in the modulation contrast imagingtechnique.

Specific Example #1

Below, a first example of the present invention that attains the firstobject of the invention is given.

FIG. 1(a) illustrates the layout of components and ray paths of theentire structure of an optical microscope that has optical modulationelements relating to the present invention. FIG. 1(b) illustrates anaperture element having a slit aperture. FIG. 1(c) illustrates acorresponding optical modulation element.

FIGS. 2(a), 2(e) and 2(g) illustrate other examples of aperture element13 and FIGS. 2(b), 2(f) and 2(h) illustrate corresponding opticalmodulation element 14, respectively pertaining to an optical microscopethat has optical modulation elements relating to the present invention.FIG. 2(d) is another example of an optical modulation element that canbe used with the aperture element illustrated in FIG. 2(a), and FIG.2(c) is a blow-up of the pie-shaped section shown in dashed lines inFIG. 2(b);

FIG. 1(b) illustrates one example of aperture element 13, and shows theregion 13a where the light passes through. On the one hand, FIG. 1(c)shows an example of an optical modulation element corresponding to theaperture 13a of FIG. 1(b) in an optical microscope pertaining to thepresent invention. This optical modulation element has a first lightabsorbing region 14a of transmittance Ta, a second light absorbingregion 14b of transmittance Tb, a first light transmitting region 14c oftransmittance Tc, and a second light transmitting region 14d oftransmittance Td, wherein the following conditions are satisfied.

    0.1<Ta<0.4

    Tb<0.01

    Tc=Td>0.9

Also, in the first light absorbing region, a phase shift is provided ofπ/2 radians in relation to the other regions.

Furthermore, the first light transmitting region 14c and the secondlight transmitting region 14d can have the same transmittance and becontiguous in certain portions as shown in FIG. 2(f). Thus, independentexistence of these two regions throughout the area of the opticalmodulation element as in FIG. 1(c) is not required. In short, in orderto obtain efficacy of the present invention, the transmitting regionsadjacent to the first light absorbing region 14a lie in a direction thatis orthogonal to the optical axis and the region adjacent the secondlight absorbing region 14b lies in approximately the same orthogonaldirection and must be a light transmitting region.

In the case where linear-shaped apertures as in FIG. 1(b) and opticalmodulation elements with asymmetric shapes in relation to the opticalaxis as in FIG. 1(c) are used, accompanying the exchanging of objectivelens 10, the optical modulation element must be rotated so that region14a on the optical modulation element(s) and aperture 13a of theaperture element are parallel. Thus, an operation must be performed thatadjusts these alignments. Also, in the case where the direction ofcontrast in the image is desired to be changed, the optical modulationelement(s) must be rotated. Thus, in such situations, the observer isusually required to perform an extremely bothersome operation.

However, when an aperture element as shown in FIG. 2(a) is used havingan aperture shaped as an angular segment of a ring that is centered onthe optical axis, and when the optical modulation element used is thatshown in FIG. 2(b), a rotating adjustment of the optical modulationelement becomes unnecessary by the observer when the objective lensesare exchanged.

In order to obtain the most suitable efficacy, there has already beenexplained that the width Wa of the first light absorbing region and thewidth Wb of the second light absorbing region should satisfy Equation(10). Concerning this, a specific example is given in relation to thecase where aperture 13a of FIG. 2(a) and optical modulation element 14of FIG. 2(b) are used. Furthermore, FIG. 2(c) illustrates, in greaterdetail, the angular segment enclosed by the dashed lines of FIG. 2(b).

The light flux from aperture 13a of FIG. 2(a) is made to have anumerical aperture in the range of 0.24-0.28. The inner radius ri andthe outer radius ro of aperture 13a of FIG. 2(a) are given by theequations below:

    ri=0.24 fc

    ro=0.28 fc

wherein, fc is the focal distance of condenser lens 9.

Usually, in order that a first light absorbing region completely coversthe conjugate image of the aperture, in comparing the above values, itis normal to set the inner radius ra of the first light absorbing region(FIG. 2(c)) as being slightly smaller than the inner radius ri of theaperture, and the outer radius rb of the first light absorbing region isset slightly larger than the outer radius ro of the aperture, whereby,

    ra=0.23 fo

    rb=0.29 fo

wherein,

fo is the focal distance of object lens 10.

Wa and Wb in equation (10) can be determined as follows.

    Wa=rb-ra=0.06 fo

    Wb=rc-rb=rc-0.29 fo

Therefore, the following condition is obtained from Equation (10).

    0.03 fo<Wb<0.12 fo

or,

    0.32 fo<rc<0.41 fo

In short, as shown in the above conditions, the width of the secondlight absorbing region is established, and by this, the most suitableefficacy is obtained.

In the case of FIG. 2(b), the position on the optical modulation element14 where the second light absorbing region 14b is arranged as shown onthe outside of the first light absorbing region 14a. As illustrated inFIG. 2(d), the second light absorbing region 14b may also be arranged onthe inside of the first light absorbing region. However, placing thesecond light absorbing region on the inside reverses the direction ofcontrast formation in the image where the same aperture, such as theaperture 13a of FIG. 2(a), is used.

Also, aperture 13a can alternatively be made to be multiple, asillustrate in FIG. 2(g), in order to increase the light quantity thatilluminates the sample, and corresponding light absorbing regions can beformed on the optical modulation element as shown in FIG. 2(h).

The present invention can also readily be used with microscopes thatview an image using reflected light, as illustrated in FIG. 3. Thecomponents in FIG. 3 are labeled with the same numbers as listed forFIG. 1, with the exception of new item 15 which is a beam splitter.

Also, as shown in FIG. 4(a), pupil plane 11 of the image-forming opticalsystem can be relayed to relayed pupil plane 11', and another opticalmodulation element can also be positioned at relayed pupil plane 11'.Moreover, different regions on a single optical modulation element canbe partitioned among two optical modulation elements and arranged atpupil planes 11 and 11'. FIGS. 4(b) and FIG. 4(c) illustrate examples ofpartitioning the light control regions illustrated in FIG. 2(b). Region14a and 14c are partitioned and arranged at pupil plane 11, and regions14b and 14d are partitioned and arranged at relayed pupil plane 11'.

Specific Example #2

A specific example for realizing the first and second objects of thepresent invention will now be described for objective lenses [1]-[4]which may be selected for viewing with the magnifications and numericalapertures (NA), as set forth below.

[1] Magnification 10×, NA 0.25

[2] Magnification 20×, NA 0.4

[3] Magnification 40×, NA 0.6

[4] Magnification 60×, NA 0.9

FIG. 5(a) and FIG. 6(a) illustrate the components and ray paths for aportion of the illuminating optical system according to the presentinvention for the case of a low magnification objective lens and a highmagnification objective lens, respectively. FIG. 5(b) and FIG. 6(b)illustrate an identical aperture that is arranged at pupil plane 8 ofthe illuminating optical system for each case of magnification.

Dashed line 16L illustrates the range of the light flux which can enterthe objective lens when the smallest numerical aperture objective lensis used. More specifically, the effective light flux is shown when theobjective lens [1] above is used. Also, dashed line 16H shows the rangeof the light flux when a high magnification objective lens is used. Morespecifically, the effective light flux is shown when the objective lens[4] above is used. As one example of apertures of FIG. 5(b) and FIG.6(b), aperture 13a is given a range of numerical aperture from 0.18-0.2.Equation (9) is satisfied since NAc=0.2 and NAo=0.25. Below are theranges that the first light absorbing region 14a should cover for eachof the objective lenses [1']-[4'] (corresponding to lenses [1]-[4]),given as a percentage relating to the numerical aperture of eachobjective lens.

[1'] About 72%-80%

[2'] About 45%-50%

[3'] About 30%-33%

[4'] About 20%-22%

FIG. 5(d) and FIG. 6(d) illustrate the ring-shaped phase shifters on theoptical modulation elements 14L and 14H, for objective lenses [1] and[4] respectively.

Therefore, a common aperture element 13 having an aperture 13a asillustrated in FIG. 5(b) and FIG. 6(b) is used for each of objectivelenses [1]-[4]. This allows the magnification to be frequently switchedwithout it being necessary to perform an exchanging operation of theapertures. In this example the magnification can vary from a range of 10times to 60 times and the numerical aperture can vary from 0.25-0.9, andthe same aperture element is used for each objective lens. Also, becausethe region on the optical modulation elements is a ring, even if theaperture is positioned to rotate about the optical axis, an adjustmentof the positions of the optical modulation elements is not necessary.Therefore, the labor of the observer is greatly reduced, and aneffective observation can be performed.

Also, since the aperture shape is an angular sector of a ring, a highefficacy of phase contrast can be obtained even when a low magnificationobjective lens is used because of the effect of the obliqueillumination.

By the use of the optical modulation elements as illustrated in FIG.2(b), observations that combine the previously mentioned phase contrastimaging technique and the modulation contrast imaging technique can beperformed simultaneously. In short, along with the operation becomingunnecessary for such things as exchange and adjustment of the apertures,there is good sensitivity provided in imaging structures of transparentsamples, and samples can be observed having a clear outline andthree-dimensional sense. In the above cases, the optical modulationelements corresponding to objective lenses [1] and [4] are shown in FIG.5(e) and FIG. 6(e), respectively.

Also, by making the relative positions, shapes, and varieties of eachregion on the optical modulation elements that are used in relation toobjective lenses of all magnifications in use to be uniform, theformation of contrast in the image becomes common for each objectivelens in use.

FIG. 5(c) and FIG. 6(c) are expanded views of the aperture 13a of FIG.5(b) and FIG. 6(b), respectively. Based on the angle θ, where θ is theangle subtended by the angular segment of a circular ring that forms theaperture, the effect of the oblique illumination changes A value of θ inthe range of approximately 60°-120° is thought to be the most suitable.When θ is smaller than 60°, the light quantity from small objects isinsufficient. This is especially the case with a high magnificationobjective lens, because the proportion of light occupying the pupil of azero-order light region becomes smaller than when a low magnificationobjective lens is used. Thus, the insufficiency of light quantity whenusing a high magnification lens becomes readily apparent. On the otherhand, when θ is greater than 120°, the oblique illumination becomesvague, and the image contrast drops.

However, in the case of a construction as that above, when there is highmagnification observation as previously mentioned, the image has atendency to become dark, and because the zero-order light also passesthrough a region of the optical modulation element that is near theoptical axis, the efficacy pertaining to the optical modulation elementis also low, and a good resolving power is difficult to obtain. However,upon arranging aperture 13a as shown in FIG. 7(b) to lie just within theoutermost perimeter of light flux 16L (i.e., most outward from theoptical axis while still being within light flux 16L), the efficacypertaining to the optical modulation element increases since theproportion of zero order light that is passed by such an aperture isgreater than when the apertures of FIG. 5(b) or FIG. 6(b) are used.Also, the resolution increases because the effect of the obliqueillumination becomes greater.

As one example, in relation to the objective lenses [1]-[4] above, anaperture of the shape of FIG. 7(b) having a numerical aperture in therange of 0.22-0.25 may be used. Below are the ranges that the firstlight absorbing region 14a or the phase-shifter should cover on theoptical modulation element shown in FIG. 2(b) for each of the abovenumbered objective lenses, given as a percentage relating to thenumerical aperture of each objective lens.

[1"] About 88-100%

[2"] About 55-63%

[3"] About 37-42%

[4"] About 24-28%

The figure modeled after the equations when the optical modulationelement of FIG. 2(b) is used with objective lens [4] is illustrated inFIG. 8(c).

In [4'] just as in [4"] above, the transmitting region of the zero-orderlight is near the center portion of the pupil in the vicinity of theoptical axis of the image-forming optical system (i.e., of the objectivelens). Because the transmitting region of the zero order light isfurther from the optical axis in [4"] than in [4'], the efficacy of theoptical modulation element is greater.

However, as for the case where an aperture such as that of FIG. 7(b) isused with some low magnification objective lenses, an optical modulationelement such as that shown in FIG. 2(b) cannot be used. This is becausethe FIG. 2(b) structure prepares light transmitting regions and lightabsorbing regions on the outsides of the regions corresponding toapertures. Therefore one can use the optical modulation element such asthat shown in FIGS. 7(c) when the lowest magnification objective lens isused.

In FIG. 7(c), 14f is a light absorbing ring region, and can provide aphase shift. Direct light from the aperture passes through this region.Region 14fo, located outward of this region, is outside the pupil of theobjective lens. As a result, no light passes through. Therefore when thetransmittance is established to be high in the region 14fi (which is onthe inside of region 14f), modulation contrast in the image can beattained because these regions become structures equivalent to thenormal modulation contrast imaging technique. Thus, the aperture elementof FIG. 7(b) may be used over the full range objective lenses whileproviding a modulation contrast imaging efficacy during use of thelowest magnification objective lens and providing both a modulationcontrast imaging efficacy and a phase contrast imaging efficacy duringuse of the other objective lenses.

While the invention has been illustrated and described with reference topreferred embodiments, the invention is not limited thereto. Instead,the scope of the invention is to be defined by the following claims andtheir legal equivalents. Further, all such modifications as would beobvious to one of ordinary skill in the art are intended to be withinthe scope of the following claims.

What is claimed is:
 1. An optical modulation element for use in an optical microscope having an optical axis, said optical modulation element including four regions comprising:a first light absorbing region having a transmittance Ta; a second light absorbing region having a transmittance Tb which is positioned adjacent to the first light absorbing region; a first light transmitting region having a transmittance Tc which is positioned adjacent to the first light absorbing region; and, a second light transmitting region having a transmittance Td which is positioned adjacent the second light absorbing region at a side of the second light absorbing region that is opposite to said first light absorbing region, wherein the following conditional equations are satisfied

    Tb<Ta<Tc

    Ta<Td

    Tc>0.5

    Td>0.5

wherein at least one of said four regions imparts a predetermined phase retardation to light transmitted therethrough.
 2. The optical modulation element according to claim 1, wherein said first light absorbing region imparts a predetermined phase retardation to light transmitted by said first light absorbing region.
 3. The optical modulation element according to claim 2 in combination with an optical microscope, said optical microscope including:an illuminating optical system; an aperture element positioned at a pupil plane of said illuminating optical system and having an aperture therein; and an image-forming optical system having an objective lens; wherein said optical modulation element is positioned at or near a pupil plane of the image-forming optical system that is conjugate to the pupil plane of said illuminating optical system.
 4. The optical modulation element according to claim 1, wherein the second light absorbing region is substantially opaque, and Tc and Td each exceed 0.90.
 5. The optical modulation element according to claim 4, wherein the first light absorbing region and the second light absorbing region are arranged along directions approximately orthogonal to the optical axis.
 6. The optical modulation element according to claim 4 in combination with an optical microscope, said optical microscope including:an illuminating optical system; an aperture member positioned at a pupil plane of said illuminating optical system and having an aperture therein; and an image-forming optical system having an objective lens; wherein said optical modulation element is positioned at or near a pupil plane of the image-forming optical system that is conjugate to the pupil plane of said illuminating optical system.
 7. The optical modulation element according to claim 1, wherein the first light absorbing region and the second light absorbing region are arranged along directions approximately orthogonal to the optical axis.
 8. The optical modulation element according to claim 2, wherein the first light absorbing region and the second light absorbing region are arranged along directions approximately orthogonal to the optical axis.
 9. The optical modulation element according to claim 8, wherein the second light absorbing region is substantially opaque, and Tc and Td each exceed 0.90.
 10. The optical modulation element according to claim 9, and further comprising the first light absorbing region and the second light absorbing region each having sides that are equidistant, with the below conditional equation being satisfied

    Wa/2<Wb<2Wa

wherein, Wa is the distance between the equidistant sides of the first light absorbing region, and Wb is the distance between the equidistant sides of the second light absorbing region.
 11. The optical modulation element according to claim 10, wherein the aperture of the aperture element is the shape of an angular segment of a ring centered on the optical axis, and the first light absorbing region and the second light absorbing region of the optical modulation element are each rings of a different size that are centered on the optical axis.
 12. The apparatus as set forth in claim 11, said aperture element being rotatable about the optical axis.
 13. The optical modulation element according to claim 11, wherein the second light absorbing region is arranged outward from the optical axis of said first light absorbing region, light passing through the innermost region of the aperture is incident on a sample being observed by said microscope, the optical microscope includes multiple objective lenses for observing an image having a selected magnification and the following conditional equation is satisfied

    NAc>0.7 NAo

wherein, NAc is the numerical aperture value subtended by the region of said aperture having the smallest angular subtense from the sample surface, and NAo is the numerical aperture of the lens having the smallest numerical aperture value of said multiple objective lenses.
 14. The apparatus according to claim 13, wherein:when the objective lens having the smallest numerical aperture value is used, a part of said first light absorbing region is positioned at a circumference region of a pupil of said image-forming optical system.
 15. The combination as recited in claim 14, said aperture element being rotatable about the optical axis.
 16. The optical modulation element according to claim 7 in combination with an optical microscope, said optical microscope including:an illuminating optical system; an aperture element positioned at a pupil plane of said illuminating optical system and having an aperture therein; and an image-forming optical system having an objective lens; wherein said optical modulation element is positioned at or near a pupil plane of the image-forming optical system that is conjugate to the pupil plane of said illuminating optical system.
 17. The optical modulation element according to claim 1 in combination with an optical microscope, said optical microscope including:an illuminating optical system; an aperture element positioned at a pupil plane of said illuminating optical system and having an aperture therein; and an image-forming optical system having an objective lens; wherein said optical modulation element is positioned at or near a pupil plane of the image-forming optical system that is conjugate to the pupil plane of said illuminating optical system.
 18. The optical modulation element according to claim 1, wherein an aperture of an aperture element is the shape of an angular segment of a ring centered on the optical axis, and the first light absorbing region and the second light absorbing region of the optical modulation element are each rings of a different size that are centered on the optical axis.
 19. The apparatus as set forth in claim 18, an aperture element being rotatable about the optical axis.
 20. The optical modulation element as set forth in claim 1, wherein Tc equals Td, and the first light transmitting region and the second light transmitting region are contiguous in certain portions of said optical modulation element. 